The invention of this application relates to a microscope system. More particularly, the invention of this application relates to a novel microscope system of high performance and function, which is able to achieve a high quality image of a high spatial resolution by illuminating a dyed specimen with lights of plural wavelengths.
In the prior art, there have been developed various types of optical microscopes, and their performance has been enhanced according to the development in the peripheral technique including the laser technique and the electronic graphic technique. As one of these high performance optical microscopes, there has been proposed (in Japanese Patent Application No. 6-329165) a microscope which is able, by using a double resonance absorption process induced by illuminating a specimen with lights of plural wavelengths, not only to control the contrast of an image to be obtained but also to perform a chemical analysis.
This optical microscope can, by using the double resonance absorption, select a specific molecule and observe absorption and fluorescence caused by a specific optical transition. First, an electron of a valence orbit 2, owned by the molecule in a ground state illustrated in FIG. 1, is excited to a valence orbit 3 that is a vacant orbit by light irradiation, as illustrated in FIG. 2. This is a first excited state. Next, the electron on a valence orbit 1 is excited, as illustrated in FIG. 3, to a hole generated on the valence orbit 2 by irradiating them with a light of another wavelength. This is a second excited state. The molecule then returns to the ground state while emitting fluorescence or phosphorescence, as illustrated in FIG. 4. And, an absorption image or a luminous image is observed by using the absorption process of FIG. 2 or the emission of the fluorescence or phosphorescence of FIG. 4.
At first, when the molecule composing a specimen is to be excited to the first excited state with a light of a resonance wavelength xcex1 by, for example, a laser beam, the number of molecules in the first excited state in a unit volume increases as an intensity of the irradiation light increases. Since a linear absorption coefficient is given as a product of an absorption cross-section per molecule and the number of molecules per unit volume, in the excitation process of FIG. 3, the linear absorption coefficient for the light of a resonance wavelength xcex2 subsequently applied depends upon the intensity of the light of the wavelength xcex1 applied first.
In short, the linear absorption coefficient for the wavelength xcex2 can be controlled with the intensity of the light of the wavelength xcex1. This indicates that, when irradiating a specimen with lights of two wavelengths xcex1 and xcex2 and obtaining a transmission image by the wavelength xcex2, contrast of the transmission image can be completely controlled with a quantity of the light of the wavelength xcex1.
On the other hand, when the deexcitation process from the second excited state of FIG. 3 by fluorescence or phosphorescence is possible, the luminous intensity is proportional to the number of molecules in the first excited state. This makes it possible to control an image contrast, even when used as a fluorescent microscope.
Further, this optical microscope of the prior art is able not only to control the contrast but also to perform the chemical analysis. Since the outermost valence orbit in FIG. 1 has an energy level intrinsic to a molecule, the wavelength xcex1 is different for the molecule. At the same time, the wavelength xcex2 is also intrinsic to the molecule. As a result, the molecule to absorb or emit a light can be restricted from the two wavelengths xcex1 and xcex2, so that an accurate chemical composition of a specimen can be identified.
Moreover, when the valence electron is to be excited, only a light having a specific electric-field vector with respect to a molecular axis is intensively absorbed. Thus, if an absorption image or fluorescence image is obtained while determining the directions of polarization of the wavelengths xcex1 and xcex2, the direction of orientation can also be identified for the same molecule.
In recent years, there has also been proposed (in Japanese Patent Application No. 8-302232) a fluorescent microscope which has a high spatial resolution exceeding a diffraction limit by using the double resonance absorption process.
FIG. 5 is a conceptional diagram illustrating the double resonance absorption process in molecule. It is illustrated in FIG. 5 that a molecule in the ground state is excited to the first excited state with the light of the wavelength xcex1 and further to the second excited state with the light of the wavelength xcex2 and that fluorescence from this second excited state is extremely weak for some kinds of a molecule.
The molecule having such optical properties experiences a remarkably interesting phenomenon. FIG. 6 illustrates an extension of a spatial distance in the double resonance absorption process, with an abscissa being an X axis. In FIG. 6, there are illustrated a spatial area A1 which is irradiated with the light of the wavelength xcex2 and a spatial area A0 which is not irradiated with the light of the wavelength xcex2. In this spatial area A0, a great number of the molecules being in the first excited state are generated by the xcex1 light excitation. At this time, fluorescence emitted with a wavelength xcex3 from the spatial area A0 can be observed. In the spatial area A1, however, the irradiation of the light of the wavelength xcex2 excites most of the molecules in the first excited state instantly to the second excited state at a higher level, so that the molecules in the first excited state disappears. As a result, the fluorescence of the wavelength xcex3 completely disappears, and further, the fluorescence from the second excited state does not exist intrinsically, so that the fluorescence itself is completely inhibited in the spatial area A1. It is therefore understood that the fluorescence exists only in the spatial area A0.
This result has a remarkably important meaning if considered from the field of application of the microscope. In the scannigng type laser microscope of the prior art, a laser beam is condensed to produce a micro beam thereby to scan a specimen to be observed. At this time, the size of the micro beam is determined by the diffraction limit which in turn is determined by a numerical aperture of a condenser lens and a wavelength, so that a higher spatial resolution cannot be expected on principle. However, according to FIG. 6, since the fluorescent area is inhibited with the irradiation of xcex2, by overlaping the wavelengths of two kinds of xcex1 and xcex2 skillfully, the fluorescent area is made narrower than the size determined by the numerical aperture of the condenser lens and the wavelength, while noticing the irradiation area of xcex1 for example. Thus, the spatial resolution is substantially improved. Therefore, by adopting this principle, it is possible to provide a fluorescent microscope exceeding the diffraction limit. This is a super-resolution microscope using the double resonance absorption process.
In order to enhance the super-resolution of this microscope, another proposal has been made (in Japanese Patent Application No. 9-25444). A molecule of various kinds, which has three quantum states including at least the ground state and in which a thermal relaxation process is more dominant than a relaxation process by fluorescence in transition upon deexcitation from a higher excited state excepting the first excited state to the ground state, is employed as a fluorescence labeler molecule. The specimen in which the fluorescence labeler molecule and a bio-molecule dyed biochemically are chemically bonded, is irradiated with the light of the wavelength xcex1 to excite the fluorescence labeler molecule to the first excited state and is then instantly excited with the light of the wavelength xcex2 to a higher quantum level, so that fluorescence from the second excited state is inhibited, thereby to inhibit the spatial fluorescent area artificially, thus the spatial resolution can be improved.
The optical properties of such molecule can be described in the following manner from the standpoint of quantum chemistry.
Generally, each atom composing a molecule is bonded by a or xcfx80bonds. In other word, according to the quantum chemistry, molecular orbits of the molecule has an a molecular orbit or a xcfx80molecular orbit, and an electron existing on these molecular orbits plays an important role to bond each atom. Of these, the electron on the a molecular orbit intensely bonds each atom to determine an inter-atomic distance in the molecule, which is the frame of the molecule. On the contrary, the electron on the xcfx80 molecular orbit makes little contribution to the bond of each atom, but is rather bound throughout the molecule by an extremely weak force.
In most cases, if the electron on the "sgr" molecular orbit is excited with a light, the inter-atomic distance of the molecule is highly changed to cause a drastic structure change including dissociation of the molecule. As a result, a kinetic energy of the atom and an energy given for the structural change by the light to the molecule are mostly transformed into a thermal energy. Because of this, an excitation energy is not consumed in fluorescence. Further, the structural change of the molecule occurs at an extremely high rate, for example in time period smaller than pico seconds, so that the fluorescence lifetime is short even if fluorescence occurs in that process. On the contrary, however, even if the electron on the "sgr" molecular orbit is excited, the structure itself of the molecule makes little change, but remains at a high quantum discrete level for a long time so that it is deexcited while releasing fluorescence in the order of nano seconds.
According to the quantum chemistry, the fact that a molecule has a xcfx80 molecular orbit and the fact that a molecule has a double bond are equivalent, and selection of a molecule having sufficient double bonds as a fluorescence labeler molecule is a necessary condition. Moreover, it has been confirmed (e.g., M. Fujii et. al., Chem. Phys. Lett. 171 (1990) 341).that, for a six-membered ring molecule such as a benzene or pyrazine among molecules having double bonds, fluorescence from the second electron excited state is extremely weak. Hence, if a molecule containing the six-membered ring such as the benzene or pyrazine is selected as a fluorescence labeler molecule, the super-resolution of the microscope can be effectively utilized because a fluorescence lifetime from the first excited state is long and because the fluorescence can be easily inhibited by an optical excitation from the first excited state to the second excited state.
Accordingly, if the specimen is dyed with such a fluorescence labeler molecule and is observed, not only its fluorescent image can be observed in a high spatial resolution, but also only the specific chemical group of a bio-specimen can be selectively dyed by adjusting the chemical group of the side chain of the fluorescence labeler molecule. Thus, even the detailed chemical composition of the specimen can be analyzed.
Generally, the double resonance absorption process occurs only when two optical wavelengths, a polarization state and the like satisfy specific conditions, so that the molecular structure can be known in a remarkable detail by using this fact. A polarization plane of a light and an orientation direction of a molecule have an intense correlation, so that the double resonance absorption process intensely occurs when each polarization plane of the lights of two wavelengths and the orientation direction of the molecule make a predetermined angle. Hence, by irradiating a specimen surface simultaneously with the two wavelength lights and by turning each polarization plane, disappearing level of fluorescence is changed, so that the information on a spatial orientation of a tissue to be observed is achieved from that change. This achievement can also be made by adjusting the lights of two wavelengths.
It is understood from the description thus far made that the optical microscope of the prior art using the double resonance absorption process has the super-resolution and the high analyzing ability.
In the super-resolution microscope using this double resonance absorption process, there have also been proposed a fluorescence labeler molecule which is capable of achieving more effective fluorescence inhibitation and a suitable timing of light-irradiation.
FIG. 7 illustrates one example of a timing at which a specimen is irradiated with two kinds of lights of wavelengths xcex1 and xcex2. As illustrated in FIG. 7, a pulse light shorter than a lifetime of the first excited state is employed. The life time of the first excited state is a time period for the fluorescence labeler molecule to emit fluorescence. And, the specimen is irradiated at first for a time period t with the light of the wavelength xcex1 and then with the light of the wavelength xcex2.
Qualitatively, the irradiation is performed at first for the time period t with the pulse light of the wavelength xcex1 sufficiently shorter than the lifetime of the fluorescence labeler molecule in the first excited state, thereby to produce a molecule in the first excited state in the observation area. Immediately after this, an area unnecessary for the observation is irradiated with the pulse light of the wavelength xcex2 sufficiently shorter than the lifetime of the first excited state, thereby to excite the molecule in the first excited state to the second excited state, thus inhibiting fluorescence.
This process can be further quantitatively explained.
Generally, when the molecule in the ground state is to be excited with the light of the wavelength xcex1 to the first excited state, the excitation process can be described by the following rate equation. Specifically: the number of molecules per unit area of the molecule dyed to a specimen is designated by N0; a photon flux of the light of the wavelength xcex1 is designated by I0;and the number of molecules in the ground state at a time t after irradiation of the light of the wavelength xcex1 is designated by N. Moreover, the lifetime of the first excited state is designated by xcfx84, and an absorption cross-section upon transition from the ground state to the first excited state by the light of the wavelength xcex1 is designated by a "sgr"01. Then, the rate equation is expressed in the following form:                                           ⅆ            N                                ⅆ            t                          =                              N            0                    ⁢                      xe2x80x83                    ⁢                      I            0                    ⁢                      xe2x80x83                    ⁢                      σ            01                    ⁢                      xe2x80x83                    ⁢                                    (                                                N                  0                                -                N                            )                        τ                                              Equation        ⁢                  xe2x80x83                ⁢        1            
If this equation is concretely solved, it is possible to determine the number of molecules n in the first excited state per unit volume at the time t after the light irradiation. That is.                     n        =                                                            N                0                            ⁢                              xe2x80x83                            ⁢                              I                0                            ⁢                              xe2x80x83                            ⁢                              σ                01                            ⁢                              xe2x80x83                            ⁢              τ                                      (                              1                +                                                      I                    0                                    ⁢                                      xe2x80x83                                    ⁢                                      σ                    01                                    ⁢                                      xe2x80x83                                    ⁢                  τ                                            )                                ·                      [                          1              -                              ⅇ                                  {                                                            -                                              (                                                                                                            I                              0                                                        ⁢                                                          xe2x80x83                                                        ⁢                                                          σ                              01                                                                                +                                                      1                            τ                                                                          )                                                              ⁢                                          xe2x80x83                                        ⁢                    t                                    }                                                      ]                                              Equation        ⁢                  xe2x80x83                ⁢        2            
wherein n=N0xe2x88x92N This Eq. 2 can be transformed into Eq. 4 by irradiation with the light of the wavelength xcex1 in such a small quantity as to satisfy the following Eq. 3:
According to Eq. 3, the value n is substantially proportional to the irradiation time t if the irradiation time of the light of the wavelength xcex1 is shorter than the lifetime of the molecule in the first excited state and if the photon flux of the light of the wavelength xcex1 is small.
Next, will be considered the case in which the molecule in the first excited state upon irradiation with the light of the wavelength xcex2 for a time period T immediately after irradiation of the light of the wavelength xcex1 is to be excited to the second excited state.
The photon flux of the light of the wavelength xcex2 is designated by I1; the number of molecules in the first excited state at the time (T+t) after irradiation with the light of the wavelength xcex1 is designated by n; and an absorption cross-section upon transition from the first excited state to the second excited state by the light of the wavelength xcex2 is designated by "sgr"12. Then, the rate equation on n is expressed in the following form:                                           ⅆ            N                                ⅆ            t                          =                                            -                              σ                12                                      ⁢                          xe2x80x83                        ⁢                          I              1                        ⁢                          xe2x80x83                        ⁢            n                    -                      n            τ                                              Equation        ⁢                  xe2x80x83                ⁢        5            
By solving this equation, the value n can be concretely determined as in the following equation when the irradiation with the light of the wavelength xcex1 is made for the time period t and is interrupted and when the irradiation with the light of the wavelength xcex2 is made immediately after the former irradiation:
n=(I0"sgr"01N0t)xc2x7exe2x88x92("sgr"12I1+1/xcfx84)Txe2x80x83xe2x80x83Equation 6
According to this Eq. 6, on the other hand, the value n is expressed for I1=0 with no irradiation of the light of the wavelength xcex2:
n=(I0"sgr"01N0t)xc2x7eT/xcfx84xe2x80x83xe2x80x83Equation 7
As a matter of fact, Eq. 6 indicates the number of molecules in the first excited state per unit volume in the area where the fluorescence is inhibited, and Eq. 7 indicates the number of molecules in the first excited state per unit volume in the area where the fluorescence is not inhibited. For a fluorescence yield "PHgr" of the molecule, the fluorescent intensity F1 from the fluorescence inhibited area and the fluorescent intensity F2 from the fluorescence not-inhibited area are given by the following Equations 8 and 9, respectively:
F1="PHgr"(I0"sgr"01N0t)xc2x7exe2x88x92("sgr"12I1+1/xcfx84)Txe2x80x83xe2x80x83Equation 8
F2="PHgr"(I0"sgr"01N0t)xc2x7eTxcfx84/xe2x80x83xe2x80x83Equation 9
A fluorescence inhibition ratio (=F1/F2) is determined to Eq. 10 from Eqs. 8 and 9:
F1/F2=exe2x88x92xcfx8412I1Txe2x80x83xe2x80x83Equation 10
Consequently, if the irradiation with the two kinds of lights of xcex1 and xcex2 is made at the timings illustrated in FIG. 7, it is possible to inhibit, at the ratio of Eq. 10, the fluorescence from the area not needed to be observed. According to Eq. 10, the fluorescence can be inhibited at an arbitrary ratio by adjusting the values I1 and T under the condition of T less than xcfx84.
FIG. 8 illustrates the timing for measuring a fluorescence intensity to be emitted from the observation area. Basically, the measuring timing of the fluorescent intensity is that the fluorescence intensity emitted from the observation area is measured for an ample time after the end of the irradiation with the light of the wavelength xcex2. At this measuring timing, the fluorescence from the observation area can be measured at an excellent S/N ratio with little fluorescence from the inhibited area.
FIGS. 9 and 10 exemplify the irradiation timing of the specimen with the two kinds of lights of the wavelengths xcex1 and xcex2 and the measuring timing of the fluorescent intensity from the observation area, respectively. Also with these timings illustrated in FIGS. 9 and 10, it is possible to realize the super-resolution microscope effectively.
In any of these timings of FIGS. 8 to 10, however, the time periods t and T have to be shorter than the time period xcfx84 (t, T less than xcfx84). This is because if t, T greater than xcfx84 on the contrary, the molecule in the first excited state is deexcited to the ground state during the irradiations with the two kinds of lights xcex1 and xcex2, thereby making the fluorescence itself from the observation area disappear. For t, Txe2x89xa7xcfx84, the irradiations of the two kinds of lights xcex1 and xcex2 could be made simultaneously, as illustrated in FIG. 11, and the intensity of the fluorescence emitted from the observation area could be simultaneously measured. In this case, however, the two kinds of excited intense lights xcex1 and xcex2 might move into the detector during the fluorescence measurement.
It is, therefore, desired that the specimen is irradiated under the condition of t, T less than xcfx84 with the two kinds of lights xcex1 and xcex2 at the timings illustrated in FIGS. 8 to 10. Although the S/N ratio is more or less degraded, the irradiations of the light xcex1 and xcex2 may also be effected at absolutely the same timing.
When the specimen is irradiated with the two kinds of lights xcex1 and xcex2 under the aforementioned conditions and at the aforementioned timings, it is necessary to measure the fluorescence emitted from the observation area immediately after the end of the irradiation by means of a detector. At that time, there are required to prepare gate signals by a commercially available general-purpose logic circuit and fetch the output electric signals of the detector in the memory of a personal computer.
Basically, as illustrated in the time charts of FIGS. 7 to 10, it raises the effect with fact that the fluorescent lifetime of the molecules to be dyed is longer than the pulse width. In the commercially available general-purpose logic circuit, however, since the switching rate is about 1 nsec, the time period xcfx84 itself is desired to exceed 1 nsecs. In other words, unless the fluorescent lifetime exceeds 1 nsec, the fluorescent phenomenon from the observation area ends before the detector and the measurement circuit become active, so that the measurements cannot be made. Thus, the fluorescence labeler molecule to dye the specimen is required to have a fluorescent lifetime exceeding 1 nsec.
On the other hand, noting the effective fluorescent area from which the signals are to be extracted, it is surely desirable that the fluorescent intensity is weaker in the fluorescence inhibition area, but it is desired from the view point of the improvement in the S/N ratio that the emission intensity of the effective fluorescence area is stronger. In short, the fluorescent intensity is measured from the time when the number of molecules in the first excited state just after the excitation with the light xcex1 is sufficient. According to the foregoing Eq. 9, the number of excited molecules is attenuated in the manner of an exponential function by the time constant which is determined by the excitation lifetime. Here, according to the characteristics of the exponential function, if the pulse widths t and T of the light are sufficiently shorter than the lifetime xcfx84 of the molecule in the first excited state, it is possible to measure fluorescence of a sufficiently strong intensity, i.e., an effective signal intensity from the molecule in the first excited state just after the excitation with the light xcex1. Especially if the time periods t and T are about one tenth of the lifetime of the molecule in the first excited state, the number of molecules in the first excited state is as many as 90% of the molecule number just after the excitation with the light xcex1, so that a sufficient signal intensity from the effective fluorescent area is achieved.
The optical microscope of the prior art thus far described has outstanding usefulness and technical priority in its super-resolution and analytical ability.
However, the optical microscope of the prior art needs the light of the wavelength xcex2 having an intensity sufficient for inhibiting the fluorescence from the first excited state by exciting a molecule from the first excited state to the second excited state (hereinafter, this light will be called the xe2x80x9cerase lightxe2x80x9d and the light of the wavelength xcex1 to excite a molecule from the ground state to the first excited state will be called the xe2x80x9cpump lightxe2x80x9d). Although the erase light has a slightly lower intensity than a high-intensity laser beam of several TW/cm2 in the laser scanning type fluorescence microscope using an unresonance two-photon absorption process, it still has a considerably strong intensity, so that it has raised a problem of influences on the bio-specimen.
This high-intensity laser is excessively strong against the biological cells of a specimen. Especially in case where a measurement for a long time is required, influence by heat reserve or absorption of multiple photons in the sample is very serious. Thus, such influence has to be minimized.
Moreover, wavelengths of the pump light and the erase light have to fall outside of the absorption band of the biological cells.
Furthermore, in order to realize a resolution as theoretically estimated, a beam of the erase light condensed on a specimen surface is required to have a zero intensity distribution where an intensity at its central portion is zero and to have an axially symmetric shape (hereinafter , this beam will be called the xe2x80x9chollow beamxe2x80x9d). This is because disturbance in the intensity distribution leads as it is to deterioration in the resolution of the microscope.
A laser is frequently used as a light source for the erase light, and in order to achieve a theoretical beam as mentioned above, it is a major premise that the laser must have a satisfactory beam profile, meaning that the beam having an intensity distribution symmetric with respect to an optical axis is desirable.
For example, a dye laser, as used as a light source in the prior art, has a beam shape which is close to triangle and an intensity distribution which is not uniform. As a result, the beam shape condensed on the specimen surface is not the expected hollow beam, but a deformed beam pattern, thereby causing deterioration in the resolution or reduction in the image quality of a microscope image. In addition, there has been proposed that the reduced image by a minute zonal aperture is used as the hollow beam. If this zonal aperture is utilized, however, it is difficult to make an optical alignment or to adjust the focal point. It thus takes a seriously long time to obtain a satisfactory image and needs a skillful technique.
Accordingly, in order to achieve the function of the super-resolution microscope sufficiently, an optical technique for solving those problems has been required.
Further, from the practical aspect, an excellent operability is also an important factor.
The microscope technique of the prior art can be applied to a number of fluorescence labeler molecules by synchronizing the light of the light source with the resonance wavelength of the pump light or the erase light of the fluorescence labeler molecule by means of a dye laser or an optical parametric oscillator (OPO).
However, in the dye laser there are problems such as a reduction in the quantity of light due to a deterioration in the dye and a frequent, troublesome dye exchange. The OPO is convenient but remarkably expensive. Moreover, the OPO is an extremely precise optical system requiring strict managements of humidity and temperature, and a nonlinear optical crystal used has a short lifetime and a high price, thereby making it a light source requiring a serious burden of maintenance and management on the user.
It is, therefore, preferable that a light source to be used has a fixed wavelength, a simple construction and a reasonable price.
In recent years, there has been developed a micro manipulation technique which can capture and move minute particles under observation of the microscope by using a laser beam. It has been earnestly desired to realize the microscope system which is enabled to have high operability and function by adding the function of the micro manipulation technique to the super-resolution microscope.
According to this micro manipulation technique, polarization is produced by condensing a high-intensity laser light to a dielectric particle such as a polyethylene particle, and then the particle can be captured and moved by attracting the particle to an area having the strongest electric field. In this technique, it is preferable to irradiate a particle with a laser beam in directions as various as possible in order to stably capture a specific particle.
However, for these laser beam irradiations in the various directions, many laser light sources and complex mirror optical systems are required, thereby making the move operation extremely difficult although the capture in one space is possible.
On the other hand, by the irradiation with the laser condensed beam of 100 MW/cm2 or more in one direction, the specific particle can be captured in one space and can be spatially moved with scanning of the beam. Nevertheless, the specimen is continuously exposed to the laser beam of a high intensity so that it is seriously damaged. This raises problems that biological cells are photosensitively killed and that a chemical change occurs due to a dissociation or photo-chemical reaction of the molecule themselves.
The invention of this application has been provided in view of the background thus far described and has an object to provide a novel microscope system which has a capability to condense an erase light for exciting a molecule in the first excited state to the second excited state with an excellent beam profile by using a simple, compact optical system and also has a high stability and operability and an excellent super-resolution. Also provided is a novel microscope system which has a micro manipulator function to capture and move specimen particles by using the erase light of a hollow beam without damaging the specimen.
In order to solve the above-especified problems, the invention of this application provides a microscope system.
According to an aspect of the present invention, a microscope system is provided which comprises an adjusted specimen and a microscope body, wherein the adjusted specimen is dyed with a molecule which has three electron states including at least a ground state and in which an excited wavelength band from the first electron excited state to the second electron excited state overlaps a fluorescent wavelength band upon deexcitation through a fluorescence process from the first electron excited state to a vibrational level in the ground state, wherein the microscope body includes: a light source for a light of a wavelength xcex1 for exciting the molecule from the ground state to the first electron excited state; a light source for a light of a wavelength xcex2 for exciting the molecule in the first electron excited state to the second or higher electron excited state; a condensing optical system for condensing the light of the wavelength xcex1 and the light of the wavelength xcex2 on the adjusted specimen; overlap means for partially overlapping the irradiation region of the light of the wavelength xcex1 and the irradiation region of the light of the wavelength xcex2 on the adjusted specimen; and an emission detector for detecting an emission upon deexcitation of the excited molecule to the ground state, and wherein a region of the emission upon deexcitation of the molecule from the first electron excited state to the ground state is inhibited by irradiating the light of the wavelength xcex1 and the light of the wavelength xcex2 through the overlap means.
According to another aspect of the present invention, a microscope system is provided which comprises an adjusted specimen and a microscope body, wherein the adjusted specimen is dyed with a molecule which has three electronic states including at least a ground state, wherein the microscope body includes: a light source for a light of a wavelength xcex1 for exciting the molecule from the ground state to the first electron excited state; a light source for a light of a wavelength xcex2 for exciting the molecule in the first electron excited state to the second or higher electron excited state; a condensing optical system for condensing the light of the wavelength xcex1 and the light of the wavelength xcex2 on the adjusted specimen; overlap means for partially overlapping the irradiation region of the light of the wavelength xcex1 and the irradiation region of the light of the wavelength xcex2 on the adjusted specimen; and an emission detector for detecting an emission upon deexcitation of the excited molecule to the ground state, wherein a region of the emission upon deexcitation of the molecule from the first electron excited state to the ground state is inhibited by irradiating the light: of the wavelength xcex1 and the light of the wavelength xcex2 through the overlap means, and wherein a beam obtained by condensing the light of the wavelength xcex2 has a phase distribution in which the phase is shifted by xcfx80 at a symmetric position with respect to an optical axis of the beam in a plane normal to the optical axis.
According to yet another aspect of the present invention, a microscope system is provided which comprises an adjusted specimen and a microscope body, wherein the adjusted specimen is dyed with a molecule which has three electron states including at least a ground state and in which an excited wavelength band from the first electron excited state to the second electron excited state overlaps a fluorescent wavelength band upon deexcitation through a fluorescence process from the first electron excited state to a vibrational level in the ground state, wherein the microscope body includes: a light source for a light of a wavelength xcex1 for exciting the molecule from the ground state to the first electron excited state; a light source for a light of a wavelength xcex2 for exciting the molecule in the first electron excited state to the second or higher electron excited state; a condensing optical system for condensing the light of the wavelength xcex1 and the light of the wavelength xcex2 on the adjusted specimen; overlap means for partially overlapping the irradiation region of the light of the wavelength xcex1 and the irradiation region of the light of the wavelength xcex2 on the adjusted specimen; and an emission detector for detecting an emission upon deexcitation of the excited molecule to the ground state, wherein a region of the emission upon deexcitation of the molecule from the first electron excited state to the ground state is inhibited by irradiating the light of the wavelength xcex1 and the light of the wavelength xcex2 through the overlap means, and wherein a beam obtained by condensing the light of the wavelength xcex2 has a phase distribution in which the phase is shifted by xcfx80 at a symmetric position with respect to an optical axis of the beam in a plane normal to the optical axis.
In the aforementioned microscope systems, according to the invention of this application:
the excitation wavelength band from the first electron excited state to the second electron excited state and the excitation wavelength band from the ground state to the first electron excited state are different;
the molecule is a molecule containing one or more of a six-membered ring;
the six-membered ring is a benzene ring or a purine base;
the molecule is a molecule containing one or more of a six-membered ring derivative;
the six-membered ring derivative is a benzene derivative or a purine derivative;
the molecule is any of a xanthene group molecule, a rhodamine group molecule, a oxazine group molecule, a cyanine group molecule, a coumarin group molecule, a oxazole group molecule, a oxadiazole group molecule and a stilbene group molecule;
the molecule is any of the following molecules: 2,2xe2x80x3-dimethyl-p-terphenyl; p-terphenyl (PTP); 3,3xe2x80x2,2xe2x80x3, 3xe2x80x2xe2x80x3-tetramethyl-p-quaterphenyl; 2,2xe2x80x2xe2x80x3-demethyl-p-quaterphenyl; 2-methyl-5-t-butyl-p-quaterphenyl; 2-(4-biphenylyl)-5-(4-t-butylphenyl)-1,3,4-oxiazole (BPBD-365); 2-(4-biphenylyl)-phenyl- 1,3,4-oxadiazole; 2,5,2xe2x80x3xe2x80x3,5xe2x80x3xe2x80x3-tetrametyl-p-quinquephenyl3,5,3xe2x80x3xe2x80x3,5xe2x80x3xe2x80x3-tetra-t-butyl-p-quinquephenyl; 2,5-diphenyloxazole; 2.5-diphenylfuran; PQP (p-quanterphenyl); 2,5-bis-(4-biphenylyl)-1,3,4-oxadiazole; p-quaterphenyl-4,4xe2x80x2xe2x80x3-disulfonic acid disodium salt; p-quaterphenyl-4,4xe2x80x2xe2x80x3-disulfonic acid dipotassium salt; 4,4xe2x80x2xe2x80x3-bis-(2-butyloctyloxy)-p-quaterphenyl; 3,5,3xe2x80x3xe2x80x3,5xe2x80x3xe2x80x3-tetra-butyl-p-sexiphenyl; 2-(1-naphthyl)-5-phenyloxazole; 2-(4-biphenylyl)-6-phenylbenzoxazotetrasulfonic acid potassium salt; 2-(4-biphenylyl)-6-phenylbenzoxazole-1,3; 4,4xe2x80x2-diphenylstilbene; [1,1xe2x80x2-biphenyl]-4-sulfonic acid, 4,4xe2x80x3,-1,2-ethene-diylbis-,dipotassium salt; 2,5-bis-(4-biphenylyl)-oxazole; 2,2xe2x80x2-([1,1xe2x80x2-biphenyl]-4,4xe2x80x2-diyldi-2,1-ethenediyl)-bis-benzenesulfonic acid disodium salt; 7-amino-4-methylcarbostyryl; 1,4-di[2-(5-phenyloxazole)]benzene; 7-hydroxy-4-methylcoumarin; p-bis(o-methylstylryl)-benzene; benzofuran, 2,2xe2x80x2-[1,1xe2x80x2-biphenyl]-4,4xe2x80x2-diyl-bis-tetrasulfonic-acid; 7-dimethylamino-4-methylquinolom-2; 7-amino-4-methylcoumarin; 2-(p-dimethylaminostyryl)-pyridylmethyl iodide; 7-diethylamonocoumarin; 7-diethylamino-4-methylcoumarin; 2,3,5,6-1H,4H-tetrahydro-8-methylquinolizino-[9,9a,1-gh]-coumarin; 7-diethylamino-4-trifluoromethylcoumarin; 7-dimethylamino-4trifluoromethylcoumarin; 7-amino-4-trifluoromethylcoumarin; 2,3,5,6-1H,4H-tetrahydroquinolizino-[9,9a,1-gh]-coumarin; 7-ethylamino-6-methyl-4-trifluoromethylcoumarin; 7-ethylamino-4-trifluoromethylcoumarin; 2,3,5,6-1H,4H-tetrahydro-9-carboethoxyquinolizino-[9,9a,1-gh]coumarin; 2,3,5,6-1H,4H-tetrahydro-9-(3-pyridyl)-quinolizino-[9,9a,1-gh]coumarin; 3-(2xe2x80x2-N-methylbenzimidazolyl)-7-N,N-diethylaminocoumarin; 2,3,5,6-1H,4H-tetrahydro-9-acetylquinolizino-[9,9a,1-gh]coumarin; N-methyl-4-frifluoromethylpiperidino-[3,2-g]-coumarin; 2-(p-dimethylaminostyryl)-benzothiazolylethyliodide; 3-(2xe2x80x2-benzimidazolyl)-7-N,N-diethylaminocoumarin; brillantsulfaflavin; 3-(2xe2x80x2-benzothiazolyl)-7diethyllaminocoumarin; 2,3,5,6-1H,4H-tetrahydro-8trifluoromethylquinolizino-[9,9a,1-gh]coumarin; 3,3xe2x80x2-diethyloxacarbocyanine iodide; 3,3xe2x80x2-dimethyl-9-ethylthiacarbocyanine iodide; disodium fluorescein (Uranin); 9-(o-carboxyphenyl)-2,7-dichloro-6-hydroxy-3H-xanthen-3-on2,7-dichlorofluorescien (Fluorescein 548); Fluorol 555 (Fluorol 7GA); o-(6-amino-3-imino-3H-xanthen-9-yl)-benzonic acid (Rhodamine 560); benzoic acid, 2-[ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl],perchlorate (Rhodamine 575); benzoic acid, 2-[ethylamino)-3-(ethylimino)-2,7-dimethyl-3X-xanthen-9-yl],ethyl ester, monohydrochloride(Rhodamine 590); 1,3xe2x80x2-diethyl-4,2xe2x80x2-quinolyloxacarbocyanine iodide; 1,1xe2x80x2-diethyl-2,2xe2x80x2-carbocyanine iodide; 2-[6-(diethylamino)-3-(ethylamino)-3H-xanthen-9-yl] benzonic acid (Rhodamine 610); ethanaminium,N-[(6-diethylamino)-9-(2,4-disulfophenyl)-3H-xanthen-3ylidene]-N-ethylhydroxide, inner salt, sodium salt; Malachit Green; 3,3xe2x80x2-diethylthiacarbocyanine iodide; 1,3xe2x80x2-diethyl-4,2xe2x80x2-quinolyloxacarbocyanine iodide; 8-(2-carboxyphenyl)2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9xe2x80x2,9a,1-hi]xantylium perchlorate (Rhodamine 640); 4-dicyanmethylene-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran; 3,3xe2x80x2diethyloxadicarbocyanine iodide; 8-(2,4-disulfophenyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a, 1-bc:9xe2x80x2, 1-hi]xanthene (Sulforhodamine 640); 5,9-diaminobenzo[a]phenoxazonium percrorate; 9-diethylamino-5H-benzo[a]phenoxazine-5-one; 5-amino-9diethylimino[a]phenoxanium perchlorate; 3-ethylamino-7-ethylimino-2,8-dimethylphenoxazine-5-ium perchlorate; 8-(trifluoromethyl)-2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a,1-bc:9xe2x80x2,9a,1-hi]perchlorate; 1-ethyl-2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridinium percholorate; Carbazine 122; 9-ethylamino-5-ethylimino-10-methyl-5H-benzo(a)phenoxazonium perchlorate; 3-diethylamino-7-diethyliminophenoxazonium perchlorate; 3-diethylthiatricarbocyanine iodide; Oxazine 750; 1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridinium perchlorate; 1,1xe2x80x2,3,3,3xe2x80x2,3xe2x80x2-hexamethylindodicarcyanine iodide; 1,1xe2x80x2-diethyl-4,4xe2x80x2-carbocyanine iodide; 2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-1,3,3-trimethyl-3H-indolium perchlorate; 2-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-3-ethylbenzothoazolium perchlorate; 1,1xe2x80x2-diethyl-2,2xe2x80x2-dicarbocyanine iodide; 1-ethyl-4-(4-(9-(2,3,6,7-tetrahydro 1H,5H-benzo(ij)-chinolinozinium))-1,3-butadienyl)-pyridinium perchlorate; 3,3xe2x80x2-dimethyloxatricarbocyanine iodide; 1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-quinolinium perchlorate; 8-cyano2,3,5,6,11,12,14,15-octahydro-1H,4H,10H,13H-diquinolizino[9,9a1-bc:9axe2x80x2, 1-hi]xanthylium perchlorate (Rhodamine 800); 2-(6-(4direthylaminophenyl)-2,4-neopentylene-1,3,5)-3-methylbenzothiazolium perchlorate; 1,1xe2x80x2,3,3,3xe2x80x2,3xe2x80x2-hexamethylindotricarbocyanine iodide; IR125; 3,3xe2x80x2-diethylthiatricarbocyanine iodide; IR144; 2-(6-(9-(2,3,6,7-tetrahydro-1H,5H-benzo(i,j)-chinolinozinium))-2,4-neopentylene-1,3,5-hexatrienyl)-3-methylbenzothiazolium perchlorate; 3,3xe2x80x2-diethyl-9,11-neopentylenethiatricarbocyanine iodide; 1,1xe2x80x2,3,3,3xe2x80x2-hexamethyl-4,4xe2x80x2,5,5xe2x80x2-dibenzo-2,2xe2x80x2-indotricarbocyanine iodide; 3,3xe2x80x2-diethyl-4,4xe2x80x2,5,5xe2x80x2-dibenzothiatricarbocyamine iodide; 1,2xe2x80x2-diethyl-4,4xe2x80x2-dicarbocyanine iodide; IR140; 2-(8-(4-p-dimethylaminophenyl)-2,4-neopentylene-1,3,5,7octatetraenyl)-3-methylbenzothiazolium perchlorate; IR 132; 2-(8-(9-(2,3,6,7-tetrahydro-1H,5H,benzo(ij)chinolinozinium))-2,4-neopentylene-1,3,5,7-octatetraenyl)-3-methylbenzothiazolium perchlorate; IR26; and IR5;
an optical axis of a beam obtained by condensing the light of the wavelength xcex1 and the optical axis of the beam obtained by condensing the light of the wavelength xcex2 are coaxial;
the beam obtained by condensing the light of the wavelength xcex2 has a phase distribution in which the phase changes continuously from 0 to 2xcfx80 when turned once around the optical axis in a plane normal to the optical axis;
the beam obtained by condensing the light of the wavelength xcex2 has a phase distribution in which the phase changes discontinuously from 0 to 2xcfx80 when turned once around the optical axis in a plane normal to the optical axis;
the beam obtained by condensing the light of the wavelength xcex2 is a Bessel beam;
the Bessel beam is a 1-st-order-Bessel-beam;
the beam obtained by condensing the light of the wavelength xcex2 is a laser beam having a vibrational mode of any of the Gauss""s type, Laguerre""s type and Hermitian""s type;
any of a gas laser, a solid laser and a semiconductor laser is provided as the light source for the light of the wavelength xcex1;
an oscillation wavelength of any of the gas laser, the solid laser and the semiconductor laser is the wavelength xcex1;
a harmonic-wave of an oscillation wavelength of any of the gas laser, the solid laser and the semiconductor laser has the wavelength xcex1;
a sum frequency of or a difference frequency between an oscillation wavelength of any of the gas laser, the solid laser and the semiconductor laser and a harmonic-wave of the oscillation wavelength has the wavelength xcex1;
any of a gas laser, a solid laser and a semiconductor laser is provided as the light source for the light of the wavelength xcex2;
an oscillation wavelength of any of the gas laser, the solid laser and the semiconductor laser is the wavelength xcex2;
a harmonic-wave of an oscillation wavelength of any of the gas laser, the solid laser and the semiconductor laser has the wavelength xcex2;
a sum frequency of or a difference frequency between an oscillation wavelength of any of the gas laser, the solid laser and the semiconductor laser and a harmonic-wave of the oscillation wavelength has the wavelength xcex2;
the gas laser is any of an excimer laser, a copper vapor laser, an argon laser, a Hexe2x80x94Ne laser, a CO2 laser, a Hexe2x80x94Cd laser and a-nitrogen laser;
the gas laser is of a mode-locked type;
the solid laser is any of a Nd:YAG laser, a Ti sapphire laser, a YLF laser and a ruby laser;
the solid laser is of a semiconductor-laser-excited type;
the solid laser is of a mode-locked type;
the microscope body has one or more of nonlinear media or wavelength modulating element for converting the wavelength of a laser beam from the gas laser, the solid laser or the semiconductor laser;
the nonlinear media or the wavelength modulating element is a nonlinear crystal;
the nonlinear media or the wavelength modulating element is a Raman shifter;
the light of the wavelength xcex1 is prepared by modulating a wavelength of a fundamental-wave of the gas laser or the solid laser with the nonlinear media or the wavelength modulating element;
the light of the wavelength xcex1 is prepared by modulating a wavelength of a harmonic-wave of the gas laser or the solid laser with the nonlinear media or the wavelength modulating element;
the light of the wavelengthxcex2 is prepared by modulating a wavelength of a fundamental-wave of the gas laser or the solid laser with the nonlinear media or the wavelength modulating element;
the light of the wavelength xcex2 is prepared by modulating a wavelength of a harmonic-wave of the gas laser or the solid laser with the nonlinear media or the wavelength modulating element;
the condensing optical system for the light of the wavelength xcex2 has a phase plate having a refractive-index distribution or an optical-path-difference distribution which gives, to a beam obtained by condensing the light of the wavelength of the xcex2, a phase difference distribution in a plane normal to an optical axis of the beam;
the condensing optical system for the light of the wavelength xcex2 has a zonal optical system;
the condensing optical system for the light of the wavelength xcex2 has a diffractive optical system;
the condensing optical system for the light of the wavelength xcex2 has an axicon;
in a resonator of the gas laser, the solid laser or the semiconductor laser, there is provided at least one of a ring-shaped zonal mirror, a zonal diffraction grating, a Fresnel zone plate, a zonal aperture, and a phase plate which gives a phase difference in which electric fields axially symmetric in a plane normal to the optical axis are shifted by x from each other;
the microscope body has an emission condensing optical system for condensing an emission from the molecule to the emission detector;
the emission condensing optical system has a sharp cut filter;
the emission condensing optical system has a notch filter;
the emission condensing optical system has a band-pass filter;
the band-pass filter transmits the emission from the molecule while not transmitting the light of the wavelength xcex1 and the light of the wavelength xcex2;
the adjusted specimen is sealed by seal means made of a substance transmitting the light of the wavelength xcex1 and the light of the wavelength xcex2;
the adjusted specimen is covered with cover means made of a substance transmitting the light of the wavelength xcex1 and the light of the wavelength xcex2;
said substance is synthetic quartz SiO2, CaF2, NaF, Na3AlF6, LiF, MgF2, SiO2, LaF3, NdF3, Al203, CeF3, PbF2, MgO, ThO2, SnO2, La2O3 or SiO;
the microscope body has a continuous-wave laser separately of the light sources for the light of the wavelength xcex1 and the light of the wavelength xcex2, and wherein a beam obtained by condensing the continuous-wave laser on the adjusted specimen has a phase distribution in which the phase is shifted by xcfx80 at a position symmetric with respect to an optical axis of the beam in a plane normal to the optical axis; and
the microscope body has means for relatively scanning, on the adjusted specimen, with a beam obtained by condensing the continuous-wave laser on the adjusted specimen, independently of the beam obtained by condensing the light of the wavelength xcex1 and the beam obtained by condensing the light of the wavelength xcex2.